Delayed-type Hypersensitivity Model Research Articles

Targeted depletion of lymphotoxin-alpha-expressing Th1 and Th17 cells inhibits autoimmune disease (48.22)

Abstract Aberrant autoimmune responses are often underpinned by dysregulated and uncontrolled CD4+ T helper (Th) cell subsets Th1 and Th17. We identify surface lymphotoxin-alpha (LTa) as common to Th1 and Th17 cells and employ a novel strategy to target these subsets using a depleting monoclonal antibody directed to surface LTa. A depleting anti-LTa mAb inhibited T cell-mediated models of delayed-type hypersensitivity and experimental autoimmune encephalomyelitis. In collagen-induced arthritis (CIA), preventive and therapeutic administration of anti-LTa; inhibited disease, resulting in immunoablation of T cells expressing IL-17, IFN-g; and TNF-a, whereas a decoy LTbR fusion protein had no effect. A mutation in the Fc tail, rendering the antibody incapable of FcgR binding and ADCC activity, abolished all in vivo effects. Efficacy in CIA was preceded by a loss of rheumatoid-associated cytokines IL-6, IL-1b and TNF-a within joints. These data indicate that depleting Th1 and Th17 with anti-LTa mAb may be beneficial in the treatment of autoimmune disease.

Functional hierarchy and relative contribution of the CD28/B7 and ICOS/B7-H2 costimulatory pathways to T cell-mediated delayed-type hypersensitivity

The CD28/B7 and ICOS/B7-H2 pathways are both critical for costimulating T cell immune responses. Deficiency in either pathway results in defective T cell activation, cytokine production and germinal center formation. However, the relative importance and contribution of each pathway towards T cell-mediated immunity is still not clear. To address this issue, we compared T cell responses of WT, CD28 knockout (KO), B7-H2 KO, and CD28-B7-H2 double KO (DKO) mice in a model of delayed-type hypersensitivity (DTH). While CD28 KO mice have partially compromised DTH response, DKO mice exhibited greatly diminished response, suggesting that the ICOS/B7-H2 pathway could partially compensate for the costimulation of DTH. Surprisingly, B7-H2 KO mice had comparable DTH response as WT mice, indicating that the ICOS/B7-H2 pathway is secondary to the CD28/B7 pathway in costimulating DTH. Interestingly, prolonging the period of sensitization could overcome the compromised DTH response in CD28 KO but not DKO mice, revealing a novel form of functional redundancy of ICOS/B7-H2 costimulation that is dependent on time to take effect. Taken together, our data reveal a functional hierarchy of the CD28/B7 and ICOS/B7-H2 pathways and delineated their relative contributions to the elicitation of a DTH response.

Selective amplification of glucocorticoid anti-inflammatory activity through synergistic multi-target action of a combination drug

IntroductionGlucocorticoids are a mainstay of anti-inflammatory therapy, but significant adverse effects ultimately limit their utility. Previous efforts to design glucocorticoid structures with an increased therapeutic window have focused on dissociating anti-inflammatory transcriptional repression from adverse effects primarily driven by transcriptional activation. An alternative to this medicinal chemistry approach is a systems biology based strategy that seeks to amplify selectively the anti-inflammatory activity of very low dose glucocorticoid in immune cells without modulating alternative cellular networks that mediate glucocorticoid toxicity.MethodsThe combination of prednisolone and the antithrombotic drug dipyridamole was profiled using in vitro and in vivo models of anti-inflammatory activity and glucocorticoid-induced adverse effects to demonstrate a dissociated activity profile.ResultsThe combination synergistically suppresses release of proinflammatory mediators, including tumour necrosis factor-α, IL-6, chemokine (C-C motif) ligand 5 (RANTES), matrix metalloproteinase-9, and others, from human peripheral blood mononuclear cells and mouse macrophages. In rat models of acute lipopolysaccharide-induced endotoxemia and delayed-type hypersensitivity, and in chronic models of collagen-induced and adjuvant-induced arthritis, the combination produced anti-inflammatory activity that required only a subtherapeutic dose of prednisolone. The immune-specific amplification of prednisolone anti-inflammatory activity by dipyridamole did not extend to glucocorticoid-mediated adverse effects, including corticosterone suppression or increased expression of tyrosine aminotransferase, in vivo after repeat dosing in rats. After 8 weeks of oral dosing in mice, treatment with the combination did not alter prednisolone-induced reduction in osteocalcin and mid-femur bone density, which are markers of steroid-induced osteoporosis. Additionally, amplification was not observed in the cellular network of corticotroph AtT-20/D16v-F2 cells in vitro, as measured by pro-opiomelanocortin expression and adrenocorticotropic hormone secretion.ConclusionsThese data suggest that the multi-target mechanism of low-dose prednisolone and dipyridamole creates a dissociated activity profile with an increased therapeutic window through cellular network selective amplification of glucocorticoid-mediated anti-inflammatory signaling.

Cutting Edge: Immunosuppressant as Adjuvant for Tolerogenic Immunization

Kang Y, Xu L, Wang B, Chen A, Zheng G. J Immunol. 2008;180(8):5172–5176 PURPOSE OF THE STUDY. Immunosuppressive agents are used frequently for the treatment of allergy, autoimmune disease, and transplant rejection but are thought to provide only temporary benefit. The authors of this study sought to determine if dexamethasone, a glucocorticoid with potent immunosuppressive properties, could promote long-term antigen-specific tolerance when administered with a peptide immunogen. METHODS. The authors used a model of delayed-type hypersensitivity (DTH) to hen ovalbumin by subcutaneously injecting ovalbumin twice during a 2-week interval into BALB/c mice. Mice with established DTH to ovalbumin were then immunized with an ovalbumin-derived MHCII-restricted peptide in the presence or absence of dexamethasone. All mice were retested for DTH at 2-week and 4- to 5-month time points after completion of immunization by injecting ovalbumin into the footpad and measuring the increase in footpad thickness. Blood-derived CD4+CD25+Foxp3+ regulatory T cells (Tregs) were quantitated by labeling peripheral white blood cells with carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulating the cultures with ovalbumin peptide, and analyzing for CFSE dilution (indicating cell division). Dendritic cells (DCs) in draining lymph nodes (LNs) were assessed for maturity by staining for CD11c, CD83, and CD86, as well as interleukin 10 (IL-10) in some experiments. Transgenic mice containing large numbers of ovalbumin-specific CD4+ T cells were studied also. A similar set of experiments was performed with nonobese-diabetic mice, a mouse model of autoimmune diabetes, by using dexamethasone and an insulin-derived MHCII-restricted peptide. Primary outcomes measured were median time to development of diabetes (as defined by glycosuria) and measurement of Treg numbers and antigen-specific proliferation. RESULTS. Treatment with dexamethasone induced long-term desensitization (as long as 4–5 months) in mice with preestablished DTH to hen ovalbumin as evidenced by a decrease in foot-pad swelling after ovalbumin challenge. This finding was accompanied by an expansion of CD4+CD25+Foxp3+ Tregs, which largely had specificity for ovalbumin. Dexamethasone, in the presence of ovalbumin, seemed to block DC maturation in draining LNs and facilitated differentiation of IL-10+ DCs. Furthermore, treatment of nonobese-diabetic mice with dexamethasone hindered development of spontaneous diabetes and was associated with the development of long-term antigen-specific persistent Tregs. CONCLUSIONS. Dexamethasone, when applied together with peptide, can promote long-term tolerance. The underlying mechanism may involve dexamethasone's effect on inhibiting DC maturation and promoting development of persistent antigen-specific Tregs. REVIEWER COMMENTS. Development of long-term tolerance, as opposed to transient desensitization, is the end goal of immunotherapy protocols, and this study suggests that glucocorticoids may be effective adjuvants in achieving this goal. The authors of this article speculated that other small-molecule immunosuppressant drugs, including cyclosporine and FK506, may also be effective for this purpose, but there is some evidence that these agents may actually promote development of allergic disease in children after solid organ transplantation.

Cellular Players and Role of Selectin Ligands in Leukocyte Recruitment in a T-Cell-Initiated Delayed-Type Hypersensitivity Reaction

Delayed-type hypersensitivity (DTH) reactions are characterized by a strong cellular infiltrate, including neutrophils, macrophages, and T lymphocytes. In all these cell types, both E- and P-selectin-dependent adhesion pathways play a significant role in recruitment into the inflamed skin. Accordingly, inhibition of selectin-mediated interactions (eg, by antibodies) results in impairment of acute DTH reactions. However, whether inhibition of a specific cell type is responsible for the anti-inflammatory effect or whether all leukocytes are affected remains unclear. To address this question, we used fucosyltransferase-VII knockout mice that lack functional selectin ligands as either donors or recipients in a DTH model elicited by Th1 cell and antigen transfer. We found that selectin-mediated adhesion is required by Th1 effector cells to enter the DTH reaction site and, additionally, to elicit the DTH reaction. On the other hand, elimination of selectin binding in the recipient's neutrophils and macrophages by use of fucosyltransferase-deficient mice receiving wild-type Th1 effector cells resulted in a strongly reduced infiltration of neutrophils and macrophages but unimpaired footpad swelling. These findings demonstrate a major role for both E- and P-selectin in the recruitment of different leukocyte cell types. However, only the presence of selectin ligands on T cells was critical for the inflammatory reaction. These findings reveal T cells as the predominant targets for selectin blockade that aim to suppress skin inflammation.

Lymphokine-activated killer (LAK) cells modulate the effects of IL-2 on a T cell-mediated immune response

The ability of LAK cells and/or IL-2 to affect the course of an established T cell response was examined in a delayed-type hypersensitivity (DTH) model. IL-2 greatly increased the magnitude of the response at 24 h, while LAK cells alone had no effect. The administration of LAK cells and IL-2 together also had no effect on the magnitude of the DTH response, demonstrating that LAK cells were able to remove the enhancement seen with IL-2 alone. The presence of LAK cells reduced the serum half-life of IL-2 significantly, but not to an extent able to account for the observed loss of IL-2 induced DTH enhancement. IL-2 administration influenced cell phenotypes in the spleen and draining lymph nodes (DLN), as well as increasing splenic weight; the additional presence of LAK cells markedly altered these effects of IL-2 in the spleen (but not the DLN). Taken together, these results suggest that LAK cells interact with activated T-cells within the immune system and modulate their function.

Helminth Products as a Potential Therapeutic Strategy for Inflammatory Diseases

Helminths secrete several molecules that can modulate the immune responses, favoring their evasion and perpetuate their survival in the host. These molecules interfere with antigen presentation, cell proliferation and activation, antibody production, cause cell death, and stimulate regulatory responses. Here, we focus on some helminth products and address their immunomodulatory effects in the host immune system and, also, we describe some anti-inflammatory properties of an Ascaris suum-derived immunomodulatory molecule, named PAS-1. This protein is a 200-kDa molecule isolated by affinity chromatography using MAIP-1 (monoclonal antibody which recognizes PAS-1), coupled to Sepharose 4B. It suppresses the inflammatory responses in murine models of delayed-type hypersensitivity, lung allergic inflammation and LPS-induced inflammation into air pouches. PAS-1 also stimulates the secretion of regulatory cytokines such as IL-10 and TGF-beta and primes IFN-gamma-secreting CD8+ and IL-10/ TGF-beta-secreting CD4+CD25+ cell clones that avoid the lung inflammation. Thus, this protein is a potent immunomodulatory component that may be used for therapeutic interventions in inflammatory diseases.

Differential responses to cyclosporine A in a delayed type hypersensitivity model

DTH is classically defined as a model of inflammation involving antigen presentation, T cell activation and cytokine production. Understanding the etiology of the reaction is important, as DTH is commonly used to assess the efficacy of reagents targeting pathways involved in inflammation. We studied the sRBC induced DTH response in two different mouse strains, the commonly used Balb/c and autoimmune prone B6D2F1. Both mouse strains exhibited equivalent paw swelling, but interestingly, CsA treatment reduced swelling in Balb/c but not in B6D2F1 mice. Restimulation of T cells from the draining lymph nodes demonstrates that Balb/c T cells produce greater levels of IFNγ, TNFα and IL-10 compared to B6D2F1 T cells, suggesting that Balb/c DTH may be more cytokine mediated than B6D2F1 DTH. Moreover, IFNγ deficient mice on the Balb/c background have reduced DTH. Although the development of autoantibodies in B6D2F1 mice has been linked to IL-17, we found that treatment with αIL-17A or αIL-17F had no effect on DTH in either strain. Our results indicate that the pathology of the DTH response is different in Balb/c compared to B6D2F1 mice, implying that different etiologies are involved. Therefore, understanding the DTH response in a particular strain of mouse is important in order to accurately determine the specific pathways responsible for the resultant inflammation.

Role of cytokines in mediating mechanical hypernociception in a model of delayed-type hypersensitivity in mice

In the present study, we used the electronic version of the von Frey test to investigate the role of cytokines (TNF-α and IL-1β) and chemokines (KC/CXCL-1) in the genesis of mechanical hypernociception during antigen-induced inflammation in mice. The nociceptive test consisted of evoking a hindpaw flexion reflex with a hand-held force transducer (electronic anesthesiometer) adapted with a 0.5 mm 2 polypropylene tip. The intraplantar administration of methylated bovine serum albumin (mBSA) in previously immunized (IM), but not in sham-immunized (SI) mice, induced mechanical hypernociception in a dose-dependent manner. Hypernociception induced by antigen was reduced in animals pretreated with IL-1ra and reparixin (a non-competitive allosteric inhibitor of CXCR2), and in TNF receptor type 1 deficient (TNFR1−/−) mice. Consistently, antigen challenge induced a time-dependent release of TNF-α, IL-1β and KC/CXCL-1 in IM, but not in SI, mice. The increase in TNF-α levels preceded the increase in IL-1β and KC/CXCL1. Antigen-induced release of IL-1β and KC/CXCL1 was reduced in TNFR1−/− mice, and TNF-α-induced hypernociception was inhibited by IL-1ra and reparixin. Hypernociception induced by IL-1β in immunized mice was inhibited by indomethacin, whereas KC/CXCL1-induced hypernociception was inhibited by indomethacin and guanethidine. Antigen-induced hypernociception was reduced by indomethacin and guanethidine and abolished by the two drugs combined. Together, these results suggest that inflammation associated with an adaptive immune response induces hypernociception that is mediated by an initial release of TNF-α, which triggers the subsequent release of IL-1β and KC/CXCL1. The latter cytokines in turn stimulate the release of the direct-acting final mediators, prostanoids and sympathetic amines.

Coptis chinensisExtract Inhibits the Production of Inflammatory Mediators and Delayed Type Hypersensitivity in Mice

Coptis chinensis rhizome has been used as a medicinal herb in traditional Oriental medicine. We investigated the effects of Coptis chinensis extract on inflammatory mediators and delayed type hypersensitivity in mice. Methods: The inhibitory effect of ethanolic extract of Coptis chinensis (CCE) on cell proliferation was evaluated using MTS assay. The lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and the Con A-activated mouse splenocytes were cultured with various concentrations of CCE. Total nitric oxide (NO) production was determined by Griess reaction. The amounts of secreted prostaglandine E2 (PGE 2 ), interleukin (IL)-2 and IFN-γ were measured by ELISA. To investigate the in vivo anti-inflammatory effect of CCE, oxazolone-induced delayed type hypersensitivity (DTH) model was used. Results: The CCE at 100μg/ml significantly blocked the LPS-induced production of pro-inflammatory mediators (NO and PGE) in RAW264.7 macrophages. Also, it significantly inhibited cell proliferation and cytokine (IL-2 and IFN-γ) production in splenocytes. Furthermore, when splenocytes from CCE fed mice (200 mg/kg for 2 weeks) were activated with Con A, cell proliferation and cytokine production were significantly inhibited. In addition, CCE decreased in vivo inflammation in oxazolone-induced DTH model mice. Conclusion: We suggest that Coptis chinensis can be used as an anti-inflammatory drug by exerting an inhibitory effect in inflammatory mediator- and cell-mediated inflammation.

In vitro and in vivo effects of Ranunculus peltatus subsp. baudotii methanol extract on models of eicosanoid production and contact dermatitis

Ranunculus (Crowfoot) species are numerous and they are all reputed to be counter-irritants and are used in several topical conditions. In order to study the pharmacological mechanisms of action underlying this popular use, a methanol extract of Ranunculus peltatus was tested in vitro in various assays involving eicosanoid and human elastase release by intact cells as well as in vivo, with models of delayed-type hypersensitivity (DTH) contact dermatitis. The extract proved to be a selective inhibitor of the cyclooxygenase-1 pathway, producing the total inhibition of 12-(S)-HHTrE release at 200 microg/mL, while leaving both 5-lipoxygenase and 12-lipoxygenase activities unaffected at the same dose. The n-hexane, chloroform and ethyl acetate fractions of the crude methanol extract inhibited LTB(4) release by intact rat peritoneal neutrophils, but more polar fractions were inactive and did not increase the 5-LOX activity as seen previously for extracts of other Ranunculus species. In the in vivo models, the methanol extract reduced the dinitrofluorobenzene (DNFB)-induced oedema by 40%, but failed to inhibit the oedema brought on by oxazolone. The results agree with the age-old assertion that Water Crowfoot species can be used as a topical antiinflammatory remedy without the prominent irritant action that accompanies the application of non-aquatic Ranunculus species.

Demethylnobiletin inhibits delayed‐type hypersensitivity reactions, human lymphocyte proliferation and cytokine production

Our aim was to examine the effect of demethylnobiletin on various experimental models of delayed-type hypersensitivity (DTH) reactions and to determine its influence on the mediators and enzymes involved in these reactions. DTH was induced in mice by oxazolone, dinitrofluorobenzene (DNFB) and sheep red blood cells (SRBC). The effect of demethylnobiletin on the ensuing DTH was studied, especially in relation to oedema formation, cell infiltration and tissue damage. Its activity on different mediators implicated in DTH reactions was also determined and its effect on nitric oxide synthase (NOS)-2 analysed. Finally, its influence on T lymphocyte proliferation, apoptosis and caspase 3 activity was tested. DTH reactions were all reduced by demethylnobiletin. The experimental results suggest that the compound may act by reducing cell infiltration and by suppressing mediators such as interleukin-2 (IC50=1.63 microM), interleukin-4 (IC50=2.76 microM), tumour necrosis factor-alpha (IC50=0.66 microM), interferon-gamma (IC50=1.35 microM), and interleukin-1 beta (46% at 2.5 microM) and by concomitantly increasing the production of the anti-inflammatory cytokine, interleukin-10. In addition, while demethylnobiletin affected nitric oxide production, it did not modify NOS-2 expression. Finally, demethylnobiletin inhibited proliferation of T cells and induced their apoptosis. Demethylnobiletin decreased DTH reactions induced by various agents. This finding, along with the fact that the compound has a low toxicity and exhibits several other interesting properties, could pave the way for other structurally related citroflavonoids to be used as pharmacological agents in complementary therapies.

GMI-1070: A Small Pan-Selectin Antagonist That Inhibits Leukocyte Adhesion and Migration in Multple Disease Models In Vivo.

Rational design of glycomimetic inhibitors based on the bioactive conformation of functional carbohydrates provides new therapeutic opportunities for the development of small molecule drugs with improved activity, pharmacokinetics, and bioavailability relative to native ligands. In designing more active glycomimetic selectin inhibitors, the low enthalpy (ΔH0) of the reaction is compensated by pre-forming the bioactive conformation thereby improving the entropy (ΔS0), known as S/H compensation. Modifications of the molecule that also stabilize the core structure, further improve ΔS0 and binding activity. To improve ΔH0, second site interactions were explored. To address the requirements for P and L-selectins, interactions were combined for both carbohydrate and sulfate-binding domains to produce heterobifunctional molecules. By stabilizing the bioactive core and exploring second site molecular interactions, we have produced a family of pan-selectin antagonists, one of which (GMI-1070) is now in development and scale-up synthesis as a lead compound. GMI-1070 is a potent inhibitor of E, P, and L-selectins in vitro and inhibits E and P-selectin-mediated leukocyte adhesion to endothelial monolayers under flow conditions. More importantly, GMI-1070 is active in several animal models of diseases requiring leukocyte adhesion and migration such as: a delayed-type hypersensitivity (DTH) response, cardiac ischemia/reperfusion injury, and vaso-occlusive crisis in sickle cell disease. GMI-1070 significantly inhibited infarct size in an ischemia/reperfusion cardiac injury model in rats. A single dose at 10mg/kg gave maximal inhibition. The effects of GMI-1070 on T-cell migration was studied in a DTH model. Mice were sensitized with oxazolone on the abdomen and then, 7 days later challenged on the ear. Donors T-cells from a different cohort of similarly sensitized mice were fluorescently labeled and injected into the test cohorts at the time of administration of GMI-1070. Three hours following injection, migration of fluorescent T-cells to the challenged area was determined. Selectin-dependent T-cell migration in the DTH response was completely eliminated with a dose of 10mg/kg of GMI-1070, suggesting the potential clinical application in diseases involving skin homing T-cells such as graft vs. host disease (GVHD) after bone marrow transplantation, and other inflammatory skin diseases. We have also shown that acute myelogenous leukemia (AML) cells adhere to endothelial cells under flow in a selectin-dependent mechanism suggesting that graft vs. leukemia as well as graft vs. host may be affected by treatment with GMI-1070. GMI-1070 was also tested in a model of vaso-occlusive crisis in sickle cell disease using Berkeley sickle cell mice. Blood flow was restored to normal values and adhesion of sickle red blood cells to adherent leukocytes was essentially eliminated as determined intravital microscopy using a dose of 20mg/kg administered at the time of elicitation of the vaso-occlusive challenge (TNFα) and at the start of intravital microscopy (70 minutes later). Based on the encouraging results in disease models, we are advancing the clinical development of GMI-1070 into Phase 1 studies to support indications which include the treatment of sickle cell patients in vaso-occlusive crisis.

Inhibitory control of endothelial galectin‐1 onin vitroandin vivolymphocyte trafficking

Galectin-1 (Gal-1) is a beta-galactoside-binding protein, the expression of which is increased in endothelial cells on exposure to proinflammatory stimuli. Through binding of several receptors (CD7, CD45, and CD43) Gal-1 is known to induce apoptosis of activated T lymphocytes, an effect thought to mediate the beneficial effects it exerts in various inflammatory models. The data presented here highlights another function for Gal-1, that of a negative regulator of T-cell recruitment to the endothelium under both physiological and pathophysiological conditions. We have shown, using siRNA to knockdown Gal-1 in endothelial cells, that endogenous Gal-1 limits T-cell capture, rolling, and adhesion to activated endothelial cells under flow. Furthermore, the reverse effect is observed when exogenous human recombinant Gal-1 is added to activated endothelial monolayers whereby a dramatic reduction in lymphocyte recruitment is seen. These findings are corroborated by studies in Gal-1 null mice in which homing of wild-type (WT) T lymphocytes is significantly increased to mesenteric lymph nodes and to the inflamed paw in a model of delayed-type hypersensitivity. In conclusion, mimicking endothelial Gal-1 actions would be a novel strategy for controlling aberrant T-cell trafficking, hence for the development of innovative anti-inflammatory therapeutics.

Effective Treatment of Inflammatory Disease Models with Exosomes Derived from Dendritic Cells Genetically Modified to Express IL-4

In this study, we demonstrate that genetically modified bone marrow-derived dendritic cells (DC) and exosomes derived from the DC, expressing either secreted IL-4 or membrane-bound IL-4, can reduce the severity and the incidence of established collagen-induced arthritis and inhibit inflammation of delayed-type hypersensitivity (DTH) in mice. The ability of the DC and DC-derived exosomes to suppress the DTH response was MHC class II and, in part, Fas ligand/Fas dependent. The DC-derived exosomes were internalized by CD11c(+) DC in the dermis at the site of injection and in the draining lymph node as well as by CD11c(+) DC and F4/80(+) macrophages in the spleen. Moreover, adoptive transfer of CD11c(+) or CD3(+) splenic cells from mice treated with exosomes showed significant reduction of footpad swelling in the DTH model. These results demonstrate that administration of DC/IL-4 or exosomes derived from DC/IL-4 are able to modulate the activity of APC and T cells in vivo through a MHC class II and partly Fas ligand/Fas-dependent mechanism, resulting in effective treatment of established collagen-induced arthritis and suppression of the DTH inflammatory response. Thus, APC-derived exosomes could be used therapeutically for the treatment of autoimmune disease and inflammatory disorders.

MHC Class II+ Exosomes in Plasma Suppress Inflammation in an Antigen-Specific and Fas Ligand/Fas-Dependent Manner

Exosomes are 50- to 100-nm vesicles that are formed within the late endocytic compartment and released from a variety of cell types. Previously, we demonstrated that exosomes derived from dendritic cells transduced with adenoviral vectors expressing IL-10, IL-4, or Fas ligand (FasL) produce anti-inflammatory exosomes able to reduce inflammation in a murine paw delayed-type hypersensitivity model, suppress the onset on murine collagen-induced arthritis, and reduce the severity of established collagen-induce arthritis. In this study, we examined the ability of endogenous, blood-borne exosomes to regulate the immune response. Exosomes isolated from plasma of mice immunized to keyhole limpet hemocyanin, but not from naive or OVA-immunized mice, were able to suppress the keyhole limpet hemocyanin-specific delayed-type hypersensitivity inflammatory response. The anti-inflammatory effect was mediated by MHC class II(+) plasma exosomes that were also FasL(+) and CD11b(+), but CD11c(-). Moreover, the anti-inflammatory effect of the MHC class II(+) plasma-derived exosomes was, in part, dependent upon the presence of FasL in the exosomes and Fas in the recipient mouse. These results suggest that exosomes in the plasma, produced by MHC class II(+) and CD11b(+) cells, have the ability to suppress the immune response in an Ag-specific manner in part through a Fas/FasL-dependent manner.

Changes of CD4+CD25+Foxp3+ regulatory T cells in aged Balb/c mice

A progressive decline in the integrity of the immune system is one of the physiologic changes during aging. The frequency of autoimmune diseases or immune disorders increases in the aging population, but the state of regulatory T (Treg) cells in aged individuals has not been well determined. In the present study, we investigated the levels, phenotypes, and function of CD4(+)CD25(+) Treg cells in Balb/c mice, which were older than 20 months. Significantly enhanced percentages of CD4(+)CD25(+) Treg cells in the periphery (blood, spleen, and lymph nodes) of the aged mice were observed. These Treg cells showed modified Vbeta family distribution, reduced levels of CD45 receptor B and CD62 ligand molecules, as well as normal levels of forkhead box p3. However, when the inhibiting function of Treg cells was assayed in the in vitro assays and in a delayed-type hypersensitivity (DTH) model, CD4(+)CD25(+) Treg cells of aged mice displayed significantly lower inhibiting ability on alloantigen-induced DTH reaction or cytokine productions (IL-2 and IFN-gamma) but not cell proliferation of effector T cells, as compared with CD4(+)CD25(+) Treg cells of young mice. In addition, the percentages of CD4(+)CD8(-)CD25(+) Treg cells in the thymi of aged mice increased significantly, but their total cell numbers decreased markedly in these mice. Our present studies indicated collectively that the percentages, phenotypes, the size of TCR repertoire, and function of CD4(+)CD25(+) Treg cells were altered significantly with aging in mice. The functional defects of CD4(+)CD25(+) Treg cells may shed light on the role of CD4(+)CD25(+) Treg cells in the increased sensitivity to autoimmune diseases of aged populations.

INHIBITING EFFECTOR MEMORY T CELLS WITH SHK(L5), A NOVEL PEPTIDE BLOCKER OF THE KV1.3 POTASSIUM CHANNEL.

Autoreactive effector memory (TEM) T lymphocytes play a major role in the pathogenesis of autoimmune diseases. Therefore, therapies that selectively target TEM cells without inducing generalized immunosuppression would have significant value. Blockers of the voltage-gated Kv1.3 K+ channel preferentially inhibit TEM cells without impairing the activity of other T cells. In proof-of-concept studies, Kv1.3 inhibitors have been shown to ameliorate disease in six different immune-mediated disorders-multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis, bone resorption following periodontitis, contact dermatitis, and delayed-type hypersensitivity (DTH)-without overt signs of toxicity. Here, we describe our progress in dissecting the mechanism by which ShK(L5), a selective Kv1.3 blocker, ameliorates autoimmune diseases. We used an adoptive DTH model system induced by the intraperitoneal injection into Lewis rats of ovalbumin-specific TEM cells engineered to express the marker gene eGFP (OVA-GFP T cells), followed by a challenge with ovalbumin in the pinna of one ear while the other ear received saline. We show that ShK(L5), injected subcutaneously at the time of challenge in the ears and every 24 hours thereafter, significantly reduced ear swelling when compared with vehicle-treated rats. In addition, ShK(L5) inhibited the proliferation of OVA-GFP T cells in vitro. We combined confocal imaging and flow cytometry to characterize the phenotype of the CD4+CD45RC-CCR7− OVA-GFP T cells both in vitro and ex vivo. Using two-photon microscopy, we showed that ShK(L5) acts by delaying the extravasation and reducing the motility of TEM cells at the site of inflammation. These findings support previous data, which show that ShK(L5) might have use for the therapy of autoimmune disorders by selectively targeting TEM cells while leaving the bulk of the immune system unimpaired.

New insight into the inhibition of the inflammatory response to experimental delayed-type hypersensitivity reactions in mice by scropolioside A

Scropolioside A, an iridoid isolated from Scrophularia auriculata ssp. pseudoauriculata, showed anti-inflammatory properties against different experimental models of delayed-type hypersensitivity. This iridoid reduced the oedema induced by oxazolone by 79% (72 h) at 0.5 mg/ear while reducing that induced by sheep red blood cells by 47% (18 h), 45% (24 h) and 36% (48 h) at 10 mg/kg. In vivo it reduced both oedema formation and cell infiltration whereas in vitro it reduced the proliferation of activated T-lymphocytes (IC 50 of 67.74 μM). Treatment with scropolioside A (100 μM) 18 and 24 h after phytohemagglutinin stimulation increased the number of cells arrested in the subG 0 phase whereas treatment 3 h after stimulation clearly increased the number of cells that passed to the S phase. Scropolioside A also inhibited the production of prostaglandin E 2, leukotriene B 4, nitric oxide, interleukin-1β, interleukin-2, interleukin-4, tumour necrosis factor-α and interferon-γ, but had no effect on the production of interleukin-10. Moreover, it modified the expression of both nitric oxide synthase-2 and cyclooxygenase-2, as well as the activation of nuclear factor-κB in RAW 264.7 macrophages.

Inhibition of the pro-inflammatory mediators’ production and anti-inflammatory effect of the iridoid scrovalentinoside

We have studied scrovalentinoside, an iridoid with anti-inflammatory properties isolated from Scrophularia auriculata ssp. pseudoauriculata, as an anti-inflammatory agent in different experimental models of delayed-type hypersensitivity. We found that scrovalentinoside reduced the edema induced by oxazolone at 0.5 mg/ear and sheep red blood cells at 10 mg/kg. The observed effect occurred during the last phase or inflammatory response; during the earlier phase or induction of the delayed-type hypersensitivity reaction, no significant activity was noted. Thus, scrovalentinoside reduced both the edema and cell infiltration in vivo and reduced lymphocyte proliferation in vitro, affecting the cycle principally during the first 48 h. Whereas cells stimulated with phytohemagglutinin changed from the G 0/G 1 phase to the S and G 2/M phases, when these same cells were treated with scrovalentinoside (100 μM), they remained in the G 0/G 1 phase. Finally, scrovalentinoside inhibited the production of the pro-inflammatory mediators’ TNF-α, IFN-γ, IL-1β, IL-2, IL-4, LTB 4, and NO, but had no effect on the production of the anti-inflammatory cytokine IL-10.